LOCUS       sequence-307866-        6047 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of C-terminally His8-Flag tagged SARS-CoV-2 
            Nsp8-Nsp7 fusion protein under control of a tetracycline-inducible 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6047)
  AUTHORS   Bresson S, Robertson N, Sani E, Turowski TW, Shchepachev V, 
            Kompauerova M, Spanos C, Helwak A, Tollervey D
  TITLE     Integrative vectors for regulated expression of SARS-CoV-2 proteins 
            implicated in RNA metabolism.
  JOURNAL   Wellcome Open Res. 2020 Nov 3;5:261. doi: 
            10.12688/wellcomeopenres.16322.1. eCollection 2020.
  PUBMED    33313418
REFERENCE   2  (bases 1 to 6047)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Wellcome 
            Open Res."; date: "2020-11-3"; volume: "5:261. doi"; pages: " 
            10.12688/wellcomeopenres.16322.1. eCollection 2020"
COMMENT     Sequence Label: pcDNA5-FRT-TO-Nsp8-Nsp7-HF_GC3opt
FEATURES             Location/Qualifiers
     source          1..6047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        111..490
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        491..694
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     645..665
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    696..714
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    717..735
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     745..769
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1775..1798
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     CDS             1811..1834
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1875..1892)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1881..2105
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2238..2257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2389..2436
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2444..3466
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2444..3466
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3596..3717
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3633..3652)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3687..3706
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3766..3782)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3766..3782)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3779..3801)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3790..3806
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3814..3820,3821..3838,3839..3844))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3859..3880
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3997..4014)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4168..4756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4248..4267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4927..5718,5719..5787))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5550..5569
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5788..5892)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5967..5986)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag
       61 ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt
      121 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
      181 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
      241 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
      301 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
      361 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
      421 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      481 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      541 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      601 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      661 gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata gagatctccc
      721 tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg cctggagacg
      781 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tccggactct
      841 agcgtttaaa cttaagcttg gtaccgagct cggatccggg atggccattg ccagcgagtt
      901 tagcagcctg cctagctacg ccgcctttgc cacagctcaa gaggcctatg aacaggccgt
      961 ggccaatggc gattctgagg tggtgctgaa gaagctgaaa aagtccctga acgtggccaa
     1021 gagcgagttc gacagagatg ccgccatgca gcggaagctg gaaaagatgg ccgaccaggc
     1081 catgacacag atgtacaagc aggccagaag cgaggacaag cgggccaaag tgacatctgc
     1141 catgcagacc atgctgttca ccatgctgag aaagctggac aacgacgccc tgaacaacat
     1201 catcaacaac gccagagatg gctgcgtgcc cctgaatatc atccctctga caaccgccgc
     1261 caagctgatg gtggtcatcc ccgactacaa cacctacaag aatacctgcg acggcaccac
     1321 cttcacctac gcctctgctc tgtgggagat ccagcaggtc gtggacgccg atagcaagat
     1381 cgtgcagctg agcgagatca gcatggacaa cagccccaat ctggcctggc ctctgattgt
     1441 gacagccctg agagccaaca gcgccgtgaa actgcaaggc tctgcaggat ctgctgccgg
     1501 ctctggcgag ttcagcaaga tgagcgacgt gaagtgcacc agcgtggtgc tgctgtctgt
     1561 tctgcagcag ctgagagtgg aaagcagcag caaactgtgg gcccagtgtg tccagctgca
     1621 caatgacatc ctgctggcca aggataccac cgaggccttc gagaagatgg tgtccctgct
     1681 gagtgtgctg ctgagcatgc aaggcgccgt ggacatcaac aagctgtgcg aggaaatgct
     1741 ggataaccgg gccacactgc aggcatcggg cggacatcac caccatcatc accatcacgc
     1801 cgcagccgca gattataaag atgacgatga caagtagctc gagtctagag ggcccgttta
     1861 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1921 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1981 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2041 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2101 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     2161 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     2221 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     2281 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     2341 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     2401 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     2461 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     2521 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     2581 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     2641 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     2701 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     2761 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     2821 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     2881 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     2941 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     3001 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     3061 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     3121 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     3181 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     3241 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     3301 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     3361 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     3421 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     3481 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     3541 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     3601 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     3661 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     3721 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     3781 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3841 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3901 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3961 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4021 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4081 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4141 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4201 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4261 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4321 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4381 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4441 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4501 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4561 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4621 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4681 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4741 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4801 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4861 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4921 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4981 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5041 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5101 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5161 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5221 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5281 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5341 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5401 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5461 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5521 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5581 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5641 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5701 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5761 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5821 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5881 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     5941 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6001 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgag
//
